Project Summary/Abstract Today it is becoming widely recognized that malignant cells very often behave differently depending on their specific tissue microenvironment (TME). Here we hypothesized Janus-faced properties of tumor TME proposing that specific patterns of TME oxygenation, extracellular pH (pHe), inorganic phosphate (Pi), redox and glutathione (GSH) homeostasis act to utilize an orchestrated mechanism to promote cancer cell survival while at the same time being highly toxic and mutagenic for normal cells. The overall goal of this project is to enable innovative magnetic resonance methods for in vivo multifunctional profiling of chemical TME to improve prediction power of early diagnostics for the malignant transition and for future rational design of TME-targeted anticancer therapeutics. The specific aims are: (SA1) To advance magnetic resonance technology and paramagnetic probes for in vivo real-time tissue microenvironment profiling. The paramagnetic probes for multifunctional measurements using Electron Paramagnetic Resonance (EPR) and Proton-Electron Double- Resonance imaging (PEDRI) techniques will be advanced to perform in vivo real-time TME profiling. Namely, trityl probes for concurrent monitoring of pO2, pHe and Pi, and nitroxide probes for pH, GSH and reducing capacity measurements will be optimized. SA 2: To perform in vivo tumor microenvironment profiling as the tumor progresses to malignancy. The array of the probes from (SA1) will be used to perform chemical TME profiling as the mammary tumors progress to malignancy using our colony of PyMT transgenic mice which spontaneously develop breast cancer and emulate human tumor staging. In addition to L-band EPR spectroscopic measurements, PEDRI functional mapping will be used to assess probe distribution and functional heterogeneity of tumor TME. Dynamic contrast-enhanced (DCE) MRI will be used to quantify tumor spatial heterogeneity. SA 3: To identify prognostic factors and evaluate efficacy of tissue microenvironment manipulation. Normalizing chemical TME may provide a new basis for anticancer TME-targeted therapeutic interventions. In SA 3A we will perform TME manipulation alone, and in combination with standard chemotherapy in SA 3B. We expect that the procedures aimed to prevent/compensate tissue hypoxia and acidosis will also affect tissue redox, Pi and GSH, and improve the outcome in a mouse model of pre-cancer breast lesions. In Aim 3B, we will test our hypothesis that TME chemical normalization will augment therapeutic effectiveness using the standard chemotherapy, docetaxel, to address the important clinical problem of tumor detoxification and chemotherapy resistance.